宫颈癌患者外周血树突状细胞的体外扩增和鉴定

 目的 探索体外扩增宫颈癌患者外周血来源的成熟树突状细胞(denelritic cell，DC)的方法，并鉴定DC的形态、结构、表型及生物学活性。方法 淋巴细胞分离液分离宫颈癌患者外周血，得到DC前体细胞(PBMC)，以(3M-CSF、IL-4、TNF-α诱导培养。用光镜和透射电镜观察形态结构，流式细胞仪检测细胞表面特异性分子，MTT法测定DC刺激T细胞增殖的活性。结果 联合应用细胞因子可诱导扩增出源自宫颈癌患者外周血的成熟DC，具有典型形态特征，较高表达HLA-DR、CD1a、CD80，能强烈激发同种异体T细胞增殖反应。结论 宫颈癌患者外周血体外培养可成功获得成熟的DC，为开展宫颈癌临床免疫治疗奠定了基础。     

肺癌患者外周血树突状细胞的生物学特性研究

目的：探讨肺癌患者外周血来源的树突状细胞（dendritic cells, DC）生物学特性。方法：从肺癌患者外周血分离获得单个核细胞，用细胞因子GMCSF (100 μg/L)，IL4 (50 μg/L)体外培养诱导。凋亡肿瘤细胞负载24 h后加入TNFα（10 μg/L）或CD40激发型单抗于培养DC中，继续诱导3～4 d。分别测定DC的表型、DC摄取抗原能力；混合淋巴细胞反应（MLR）对T细胞增殖能力的测定；细胞计数和3HTdR掺入观察DC对T细胞的激发和扩增效应，并与健康志愿者外周血来源的DC进行比较。结果： 患者外周血单个核细胞和正常人外周血单个核细胞来源的DC均高表达CD1α，CD83，CD80，CD86和HLADR等DC的相关抗原和共刺激分子；患者的未成熟DC能有效摄取FITCDextran，经TNFα或CD40激发型单抗激发诱导后，成为成熟和有功能的DC,几乎完全失去对抗原的摄取能力，与健康人外周血来源DC组相比无明显差别(P>0.05)；患者单个核细胞来源的DC在体外具有激发自体和同种异体外周血T细胞增殖的能力。结论：肺癌患者的外周血来源单个核细胞可以诱导成为具有功能的成熟DC。     

冻融人树突状细胞基因疫苗体外抗肿瘤免疫效应

目的：观察人外周血和人脐血来源的冻融树突状细胞（dendritic cell,DC）基因疫苗的形态、表型及体外诱导的CTL抗肿瘤活性。方法：细胞因子扩增人外周血和脐血DC,分别将EBV-LMP2、HPV16E6基因转染2种来源的冻融DC制备疫苗。动态形态学观察和流式细胞术检测疫苗表面分子表达,体外诱导并测定CTL活性。结果：人外周血和脐血冻融DC疫苗均高表达CD80、CD86和CD83,低表达CD14,高表达CD1a,体外均能诱导高效的CTL活性（P=0.001）,并与新鲜DC疫苗差异无统计学意义,P=0.138。结论：负载肿瘤相关病毒抗原基因的冻融DC疫苗保持了功能成熟DC的形态特征,且能诱导高效的特异性抗肿瘤免疫应答。     

人脐血树突状细胞抗神经胶质瘤的体外免疫效应研究

目的　研究肿瘤抗原致敏的树突状细胞 (DC)对神经胶质瘤细胞的免疫杀伤效应。方法　应用免疫磁珠分选脐血 CD34细胞 ,经 SCF FL3 GM- CSF IL- 4 TNF- α的联合诱导培养 DC,采用相差显微镜观察树突状细胞的形态 ;流式细胞仪作 DC的表面标志检测 ;MTT比色法测定同种异型的混合淋巴细胞反应能力和诱导CTL 毒性的检测。结果　 (1)脐血 CD34细胞在体外经细胞因子联合刺激后呈典型的 DC形态 ;(2 )流式检测 CD4 0、CD80和 CD86等成熟 DC特异性表面标志呈高表达 ,分别与诱导前比较 ,差异均有显著性 ;(3) MTT法测得脐血来源的 DC较外周血 DC刺激同种异体 T淋巴细胞增殖能力弱 ;经肿瘤抗原负载的 DC体外诱导出较强 CTL 毒性。结论　脐血 CD34细胞经体外扩增诱导的 DC具有典型的 CTL 毒性 ,为临床应用脐血来源 DC抗神经胶质瘤的生物治疗提供了理论依据     

人脐血来源树突状细胞的体外培养及鉴定

目的建立脐血来源树突状细胞（dendritic cells，DC）的体外培养方法，为进一步研究DC的功能、特性及临床应用提供了技术方法。方法取正常人脐血，分离获得单个核细胞，利用贴壁法去除悬浮细胞后，加入1000U／mL rhGM-CSF、500U／mL rhlL-4，至d7再加入500U／mLTNF-α进行培养，收集培养至d10的DC，分别从形态学、免疫表型和功能上加以鉴定。结果体外培养的d10，大量细胞出现典型的树突状形态；经流式细胞仪检测，高表达HLA-DR、CD80、CD83、CD1a、CD11c；混合淋巴细胞反应显示其在体外能有效刺激同种淋巴细胞增殖。结论在合适的细胞因子组合下，能够在体外利用脐血成功诱导出成熟DC。     

附子多糖诱导肝癌患者外周血树突状细胞分化成熟的实验研究

  目的  探讨附子多糖是否能在体外诱导肝癌患者外周血单核细胞向树突状细胞分化并成熟表达细胞表面分子, 为进一步研究附子多糖抗肿瘤作用机制奠定基础。  方法  取肝癌患者外周血体外分离单个核细胞, 去除淋巴细胞后进行培养, 实验组加入低、中、高3种不同浓度的附子多糖, 另设加入GM-CSF、IL-4和TNF-α的阳性对照组和不加任何诱导剂的阴性对照组, 共培养10d。其间进行细胞计数、倒置显微镜下动态观察细胞形态、MTT法细胞活力检测、扫描电镜细胞成像、流式细胞仪检测细胞表型(CD80、CD83、CD86)等检测。  结果  中浓度附子多糖组能够诱导肝癌患者外周血单核细胞分化为树突状细胞, 并促进细胞增殖, 高度表达CD80、CD83、CD86等共刺激分子和表面标记物, 与阳性对照组相比, 两组结果比较无明显差异, 与高、低浓度附子多糖组和阴性对照组结果相比有显著差异。  结论  适当浓度附子多糖能够在体外有效诱导肝癌患者外周血单核细胞分化为树突状细胞并表达成熟表型, 从而作为第二信号活化T淋巴细胞, 激发肿瘤免疫。      

人外周血树突状细胞诱导及免疫学特性研究

[目的]从人外周血分离、纯化、扩增树突状细胞(DC),并对其形态学和免疫学特性进行初步探讨.[方法]从人外周血分离DC前体细胞(主要为CD14 细胞)用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和重组人白细胞介素-4(rhIL-4)联合培养,诱导扩增成熟DC.观察DC形态、分析DC表型、核型及检测DC激发同种异体淋巴细胞增殖能力.[结果]分离的DC前体经rhGM-CSF和rhIL-4共同培养1周后,可获得大量成熟DC,扩增了24.5倍,纯度达90%以上.DC高表达分化抗原CD86、CD40、HLA-DR、CD83、CDIa,能强烈激活同种异体T淋巴细胞增殖.[结论]人外周血CD14 细胞经体外诱导培养,可以生成大量功能成熟的DC,从而为进一步开展DC的基础研究和临床应用打下基础.     

肺癌细胞和蛙皮素抑制树突状细胞的产生和功能

目的：了解肺癌细胞株及蛙皮素对树突状细胞（DC）产生及功能的影响。方法：树突状细胞由健康人外周血单个核细胞CD14^ ，在完全细胞培养液中加入1000U／ml GM-CSF和1000U／ml IL-4,培养7天获得，肺癌细胞株CRL－5815，CRL－5826，Bombesin和Bombesin受体拮抗剂加入培养液中，Annxin V检测DC凋亡；流式细胞仪检测CD40，CD86，CD83，CD80，HLA－DR阳性表达。结果：培养7－10天后的DC前体细胞表达高水平的HLA－DR CD80 CD86 CD83 CD40。肺癌及蛙皮素可导致DC前体细胞凋亡，而蛙皮素受体拮抗剂可部分保护蛙皮素致DC凋亡的作用。加入肺癌细胞株或蛙皮素与DC共培养时明显抑制上述细胞表型的表达和DC刺激同种异体T细胞的增殖能力，当加入蛙皮素受体拮抗剂时，DC细胞表达HLA－DR CD80 CD86 CD83 CD40明显增加，接近DC正常对照组。结论：肺癌细胞株及蛙皮素可导致DC的凋亡，抑制树突状细胞的产生和功能。     

连续贴壁法分离培养人外周血来源树突状细胞及其超微结构的观察

目的改进人外周血单个核细胞分离培养树突状细胞的方法,电镜观察树突状细胞超微结构。方法利用连续贴壁法分离获得CD14 前体细胞,经人重组粒细胞/巨噬细胞集落刺激因子(rhGM-CSF)、白介素-4(rhIL-4)和肿瘤坏死因子α(TNF-α)诱导产生成熟DC,流式细胞仪检测DC表面标志物表达水平,透射电镜(TEM)观察树突状细胞超微结构。结果连续贴壁法体外分离培养人外周血DC,所获得DC数量和纯度均都多于以往一次贴壁分离培养的DC。培养1周的DC高表达CD1a、CD40、HLA-DR、CD80、CD86。透射电镜观察到不同状态树突状细胞的超微结构。结论连续贴壁法可分离培养数量较大、纯度较高的人外周血来源DC。     

小鼠骨髓树突状细胞体外培养扩增与生物学特性研究

目的：建立一种简便高效的体外扩增培养小鼠骨髓树突状细胞（bonemarrowdendriticcell,BMDC）的方法,为树突状细胞（dendl’iticcells,DCs）的理论研究与临床应用提供实验工具。方法：联合应用重组小鼠粒细胞一巨噬细胞集落刺激网子（rmGM—CSF）、蘑纽小鼠白介素（rmlL-4）诱导培养小鼠骨髓单个核细胞,终末加入重组小鼠肿瘤坏死因子（rmTNF—α）,从形态学表型及功能方面进行检测。结果：培养3天后可见大量贴壁细胞及细胞集落形成,第5天,可见典型的树突状突起,光镜下动态观察树突状细胞DC的形态变化,流式细胞仪检测各组DC加入TNF—α前后表面标志物CD11C、CD86的衷达,加入TNF—α后CD86的阳性表达率明显增高,CD11C的阳性表达率变化无统计学意义。结论：此方法能在体外诱导和扩增出大量骨髓源性DC,为抗肿瘤疫苗研究及临床应用奠定基础。     

肝癌患者外周血树突状细胞的体外扩增、鉴定及内吞能力观察

目的　从肝癌患者外周血培养扩增树突状细胞，观察其形态、表型、内吞及递呈抗原能力，为树突状细胞在肝癌生物治疗中的应用奠定基础。方法　从肝癌患者外周血中分离单个核细胞，在 Ｇ Ｍ Ｃ Ｓ Ｆ、 Ｉ Ｌ４ 的诱导下培养扩增树突状细胞。光学显微镜下观察其体外培养过程中的形态特征及变化，电镜观察其超微结构， Ｆ Ａ Ｃ Ｓ观察其表型特征， Ｍ Ｌ Ｒ检测其抗原递呈能力， Ｈ Ｒ Ｐ内吞实验检测其群体内吞能力。结果　肝癌患者外周血树突状细胞具典型的形态及表型特征，较巨噬细胞更强的激发同种混合淋巴细胞反应的能力。其群体的内吞能力约在培养第 ５ 天达高峰，之后有明显下降。结论　从肝癌患者外周血中可获取较大量的、高纯度的、具较强抗原递呈能力的树突状细胞，在其具有活跃的内吞能力时，以合适的肿瘤抗原致敏可望成为肝癌生物治疗的崭新手段。     

晚期非小细胞肺癌患者DC-CIK治疗前的免疫状态与预后的关系

目的探讨晚期非小细胞肺癌(NSCLC)患者树突状细胞-细胞因子诱导的杀伤细胞(dendritic cellcytokine induced killers,DC-CIK)治疗前外周血CD4+CD25+CD127-调节性T细胞(regulatory T cells,TRegs)、CD3+CD56+细胞因子诱导的杀伤细胞(cytokine induced killer cells,CIKs)、CD4+/CD8+T细胞比值与预后的关系。方法采用流式细胞仪技术检测45例晚期NSCLC患者DC-CIK治疗前、后外周血CD4+CD25+CD127-TRegs、CD3+CD56+CIKs、CD4+和CD8+T细胞,并与患者临床特征进行相关分析;采用Cox回归模型分析治疗前CD4+CD25+CD127-TRegs、CD3+CD56+CIKs、CD4+/CD8+T细胞比值对患者预后的影响。结果晚期NSCLC患者DC-CIK治疗前、后比较,治疗后患者外周血CD4+T细胞减少,CD8+T细胞增多,CD4+/CD8+T细胞比值下降,差异均有统计学意义(均P<0.05);原发病灶瘤体最长直径越大,患者治疗前外周血TRegs越多,CIKs越少(均P<0.05);生存分析显示,治疗前TRegs越多,患者预后越差,生存期越短,且TRegs>7×109/L影响患者的生存(P<0.05)。结论晚期NSCLC患者外周血TRegs是判断预后的独立预测指标。     

Autologous high-killing cytotoxic T lymphocytes against human lung cancer are induced using interleukin (IL)-1beta, IL-2, IL-4, and IL-6: possible involvement of dendritic cells.

Although CTLs bear main immune responses in human tumors, stable CTL clones against human lung cancer have rarely been generated. Our previous study demonstrated efficient autologous CTL induction in human gastric cancer and glioblastoma by cytokine combination of interleukin (IL)-1beta (167 IU/ml), IL-2 (67 IU/ml), IL-4 (67 IU/ml), and IL-6 (134 IU/ml). In this study, we demonstrated successful induction of autologous stable CTLs in five of six patients with lung adenocarcinoma from mixed-lymphocyte tumor culture using this cytokine combination. All CTLs revealed potent and specific killing activity against autologous target cells (over 75% in CD8+ CTLs and over 50% in CD4+ CTLs at an E:T ratio of 10 for 24 h). Using a series of antibodies, CD8+ CTLs showed to recognize tumor-specific antigens of lung cancer cells through HLA class I. In the separate experiments, failure of CTL induction from monocyte-depleted peripheral blood mononuclear cells and appearance of cells with characteristics of dendritic cells from adherent peripheral blood mononuclear cells in the culture of the same concentration of IL-1beta, IL-4, and IL-6 indicated that CTLs can be efficiently generated by this cytokine combination via possible dendritic cell induction. This is the first study of an efficient and reproducible in vitro CTL induction against human lung cancer.     

Human renal-cell carcinoma tissue contains dendritic cells

Immune surveillance of cancer requires antigen-presenting cells which activate T cells specific for tumor-associated antigens. We show here that substantial numbers of dendritic cells, which are the most potent antigen-presenting cells, emigrate from renal-tumor explants in organ culture. Tumor-derived dendritic cells presented with all characteristics of mature dendritic cells. Dendritic cells could be identified by typical cytoplasmic projections (=veils). They expressed high levels of MHC products and of the co-stimulator CD86 (B7-2). Dendritic cells expressed the CD45RO isoform but not CD45RA. The most important point was that up to 9% of the emigrating leukocytes expressed the CD83 antigen, a specific marker for mature dendritic cells. CD83⁺ cells were approximately 40-fold enriched in the tumor tissue as compared to the peripheral blood. In contrast to cultured blood dendritic cells, tumor-emigrant dendritic cells had a reduced potential to capture soluble antigen, as shown by the exclusion of fluoresceinated Dextran molecules. Finally, in mixed leukocyte reactions, tumor-derived dendritic cells were able to stimulate naive T cells from cord blood, which is a unique feature of dendritic cells. This study demonstrates that genuine dendritic cells reside in or infiltrate renal-cell carcinoma tissue. The failure of patients with renal-cell carcinoma to mount an anti-tumor immune response despite the presence of professional antigen-presenting cells in the tumor tissue suggests that tumor-associated dendritic cells are suppressed in situ, in a similar way to that described for tumor-infiltrating lymphocytes. © 1996 Wiley-Liss, Inc.     

Immunological directives for biotherapy improvement in the treatment of colorectal cancer

A major cause of failure in biotherapy in cancer may be the non-existence of predictive indices to individualize the substances which might be helpful to switch the patients' immune response to tumour from an non-productive to a productive one. The defect in the patients' immune system needs to be identified and so the evaluation of their responses to biotherapeutic agents, in correlation to the disease progression is essential. We have addressed this problem in colorectal cancer at systemic level by examining the proliferative response of peripheral blood mononuclear cells (PBMC) to interleukins (IL) IL-2, IL-4, antiCD3 monoclonal antibody (antiCD3), IL-2+CD3, IL-2+IL-4, IL-4+CD3, IL-2+CD3+IL-4; the PBMC expression of phenotypic antigens CD3, CD4, CD8, CD25, DR, CD16, CD56, CD57 and CD19 in the patients and healthy subjects as control group. Analysing our data, it seems that as the disease progresses in these patients the peripheral blood cells change their ability to respond to activation agents which appears to be due to a phenotypic modification of their subsets. Our overall results might give a possible explanation of the variable responses to biotherapy in colorectal cancer patients.     

肺癌患者外周血及胸腔积液调节性T细胞的表达及其临床意义

目的探讨肺癌患者胸腔积液及外周血CD＋4CD＋25调节T细胞、T细胞亚群的特点及其临床意义。方法采用流式细胞术检测68例肺癌患者外周血和其中32例并发胸腔积液患者的胸腔积液及56名健康对照者外周血中CD＋4CD＋25T细胞和淋巴细胞亚群的水平。结果不同TNM分期肺癌患者外周血T淋巴细胞亚群表达比例不同,其中Ⅰ＋Ⅱ、Ⅲ、Ⅳ期患者外周血CDIcD击细胞占cD刍细胞比例分别为（19．52±3．32）％、（27．28±8．26）％、（32．31±15．60）％,均高于健康对照组的（11．12±3．32）％,差异均有统计学意义（t=31．0040、-7．9688、-4．9770,均P〈005）。合并胸腔积液患者胸腔积液CDICD嘉细胞占CD＋34细胞比例为（34．12±18．63）％,高于其外周血的（26．36±16．25）％,差异有统计学意义（t=21．164,P〈0．05）;合并胸腔积液肺癌患者胸腔积液及外周血CD＋4细胞比例分别为（25．32±13．45）％及（34．68±12．34）％,低于健康对照组的（43．24±8．68）％（t=7．3104、4．8818,均P〈0．05）,CD＋56细胞比例分别为（8．24±7．38）％及（11．23±7．65）％,低于健康对照组的（18．23±9．23）％（t=-14．7549、-11．7216,均P〈0．05）,CD＋4／CD＋8分别为（1．02±0．56）％及（1．32±0．82）％,低于健康对照组的（1．89±0．32）％,差异均有统计学意义（CD＋4／CD＋8;t=-24．78、-4．4564,均19〈0．05）。结论肺癌患者胸腔积液及外周血CD＋4CD＋25T细胞水平升高,与肿瘤患者免疫功能低下及肿瘤的发生、发展密切相关。     

凋亡肿瘤细胞致敏的树突状细胞疫苗治疗肺癌的实验研究

目的：用凋亡肿瘤细胞致敏的树突状细胞（dendritic cells，DCs）激发肿瘤抗原特异性的细胞毒T细胞（cytotoxic T lymphocytes，CTL）活性，观察其体内外抗肺癌的特性。方法：常规方法从健康人外周血单个核细胞中诱导DCs，采用或不采用凋亡肿瘤细胞负载DCs，并利用激发型CD40单克隆抗体（CD40mAb）诱导DCs成熟；成熟DCs与自体T细胞共育，分别获得Ag-CTL及non-Ag-CTL，流式细胞仪检测Ag-CTL细胞表型的变化；^3H-TdR掺入法测定DNA片段形成率；建立人肺癌细胞株A549荷瘤裸鼠模型，过继回输Ag-CTL和non-Ag-CTL，评价其在体内的抗肿瘤活性。结果：CD40mAb激发可使DCs上调CD1a、CD80、CD86、CD83、HLR-DR的表达；凋亡肿瘤细胞负载联合CD40mAb激发可进一步促进DCs的成熟；成熟DCs和自体的T细胞共育活化后CD8^＋T细胞明显上调；Ag—CTL对A549具有高效特异的杀伤力，明显强干Ag-CTL对肝癌细胞株HepG2的作用（P〈0．01），且Ag-CTL对A549的杀伤力明显强于non—Ag-CTL（P〈0．01），而non-Ag-CTL对A549及HepG2细胞的杀伤力无显著性差异；体内实验表明，Ag-CTL可有效抑制裸鼠皮下移植瘤的生长，与生理盐水组（NS组）、non-Ag-CTL组相比在统计学意义上有显著差异（P〈0．05），non-Ag-CTL组与NS组相比在统计学意义上有差异（P〈0．05）。结论：凋亡肿瘤细胞致敏的树突状细胞疫苗激发的Ag—CTL在体内外均呈现抑制肺癌细胞的特性。     

Calprotectin expression and mononuclear phagocyte subpopulations in peripheral blood and bronchoalveolar lavage

BACKGROUND AND AIM OF THE WORK: The phenotype of human alveolar macrophages (AM) can be affected by the process of maturation/differentiation and by multiple factors from the local environment. The aim of our study was to assess the expression of selected phenotypic markers characteristic for subsets of mononuclear phagocytes in bronchoalveolar lavage fluid (BAL) and peripheral blood with special attention to calprotectin (27E10), a marker of acute inflammatory macrophages. METHODS: The expression of calprotectin and 13 other phenotypic markers was evaluated by an immunoperoxidase slide assay and computer image analysis. RESULTS: We consider calprotectin (27E10 antigen) to be a marker of freshly recruited, monocyte-like, mononuclear phagocytes, being expressed in 84 +/- 13% PBM and only 10 +/- 11% of AM, p < 0.001. Computer image analysis confirmed that calprotectin-positive mononuclear cells in peripheral blood and BAL are morphologically very similar in contrast to the much larger calprotectin-negative AM. On the other hand, 25F9 antigen, the transferrin receptor (CD71), KiM8 (CD68), RFD1 (marker of dendritic cells), RFD7 (marker of mature macrophages), RFD9 (marker of epithelioid cells and macrophages of germinal centers), and RM3/1 (macrophages of late phase inflammation) were restricted preferentially to mature AM. CONCLUSIONS: Our study demonstrates phenotypic differences between mononuclear phagocytes derived from BAL and their peripheral blood precursors, and indicates markers useful for assessing the stages of maturation/differentiation of these cells. The percentage of calprotectin (27E10) positive AM might represent a parameter for assessing mononuclear phagocyte influx from peripheral blood to the lung in the very early stage of inflammatory reactions.     

Efficient generation of dendritic cells from alveolar and pleural macrophages as well as blood monocytes in patients with lung cancer.

In this study, we investigated the generation of dendritic cells (DCs) from blood monocytes and mature macrophages from untreated primary lung cancer patients. Blood monocytes were separated by adherence from blood mononuclear cells (MNC) from ten lung cancer patients and ten control subjects, and cultured for 7 days in medium with granulocyte/macrophage colony-stimulating factor (GM-CSF) plus interleukin (IL-) 4. In all cases examined, DCs with typical characteristics were obtained even in lung cancer patients after 7 days culture with these cytokines, and there was no significant difference in phenotype and stimulatory activity in allogeneic lymphocyte proliferation between DCs derived from monocytes from lung cancer patients and those from control subjects. Next, we examined whether alveolar and pleural macrophages in malignant pleural effusion separated by magnetic beads could differentiate to immunostimulatory DCs. Conventional culture conditions with GM-CSF and IL-4 did not induce efficient numbers of DCs from mature macrophages, whereas the addition of tumor necrosis factor-alpha (TNF-alpha) to GM-CSF and IL-4 effectively contributed to generate DCs. These findings suggest that both mature macrophages and blood monocytes from lung cancer patients could differentiate to DCs, and might be a useful source of DCs for immunotherapy.